JP4839254B2 - Mass spectrometry data analysis method - Google Patents

Description

The present invention relates to a mass spectrometry data analysis method. For example, the present invention relates to a mass spectrometry data analysis method that is particularly suitable for determining the valence of a target component from an MS ⁿ spectrum obtained from a liquid chromatograph mass spectrometer.

Conventionally, for example, as disclosed in Japanese Patent Application Laid-Open No. 2005-91344, it is disclosed to perform valence determination of a parent mass that is a target when performing MS ^{n + 1} analysis while performing mass analysis in real time. .

JP 2005-91344 A

In the above prior art, when acquiring data with a mass spectrometer, the valence of MS ⁿ spectrum data is determined in real time, the target MS ⁿ spectrum is selected, and then MS ^{n + 1} analysis is performed.

However, when MS ⁿ spectra derived from a plurality of components are adjacently mixed, there is a possibility that the MS ⁿ spectra derived from the plurality of components are simultaneously analyzed by MS ^{n + 1} . As a result, an MS ^{n + 1} spectrum in which a plurality of components are mixed may be detected, and the spectrum analysis may be complicated.

One object of the present invention is to provide more reliable data by re-determining the valence of the MS ^{n + 1} (n is an integer of n ≧ 1) spectrum and the MS ⁿ spectrum. .

One feature of the present invention is that, when MS ⁿ spectrum valence determination is performed on MS ⁿ (n is an integer of n ≧ 1) spectrum data acquired by a mass spectrometer, a plurality of mixed components are separated, and the valence is determined. This is a mass spectrometry data analysis method for re-determination.

  According to the present invention, more reliable data can be obtained by performing valence re-determination of isotope spectra in a region where spectra derived from a plurality of components that have been complicated in valence determination so far are mixed. It is possible to provide.

As a specific example, MS ⁿ data and MS ^{n + 1} data acquired by a mass spectrometer are subjected to valence determination by a data processing unit. Here, a region where MS spectra derived from a plurality of components are mixed adjacently is extracted, and a region where the valence is determined again is determined. Next, in order to extract at least three or more scans of MS ⁿ data and MS ^{n + 1} data within the extracted range, and to calculate the difference for each scan, between all the spectra of the first scan to the second scan Then, the difference in spectral intensity for each same mass number is obtained. Similarly, the difference in spectral intensity for each same mass number is obtained between all the spectra in the second scan to the third scan. Ask. Group the spectrum groups where the variation of the difference for each spectrum intensity found here belongs to either the increasing direction or the decreasing direction, or group the spectrum groups whose ratio variation for each spectrum intensity is within a certain allowable range. Turn into. Alternatively, both the difference and the change in the ratio are obtained, and the spectrum groups in which the difference increases and decreases in the same direction and the change in the ratio is within a certain allowable range are grouped. At this time, all scans obtained by extracting the intensities of the spectrum groups used for grouping are integrated, and the valence is determined again based on the integrated spectrum groups. Further, the fluctuation value obtained in the MS ^{n + 1} data is compared with the fluctuation value of only the MS ⁿ data in the extraction range obtained in the same procedure, and the same MS fluctuation is obtained for the target MS ⁿ spectrum and the corresponding fluctuation value. The MS ^{n + 1} spectrum is derived from the MS ⁿ spectrum. The same mass number is within a range of ± 0.1 m / z (m is a mass number and z is a charge), and the constant allowable range is usually an error range of about 0 to 25%. desirable. Hereinafter, an embodiment of the present invention will be described with reference to the drawings.

FIG. 1 is an outline of the flow of the entire liquid chromatograph mass spectrometry method used in the examples of the present invention. In the present embodiment, the sample 1 is separated by the liquid chromatograph 2 using the sample 1 which is a peptide mixed sample typified by the enzyme digested protein. A process of ionizing the separated sample with the ion source 3 is performed. A process of mass analyzing the ions introduced from the ion source by the mass analyzer 4 is performed. A liquid chromatograph mass spectrometer equipped with a detection unit 5 for detecting ions is used, and the data processing unit 6 performs processing for analyzing the acquired MS data. Here, mass analysis of the ionized sample as it is is referred to as MS ¹ analysis. Further, MS ^{n + 1} analysis is performed by selecting MS ⁿ ions as targets in the mass analyzer 4 and dissociating into MS ^{n + 1} stages using a dissociable mass spectrometer. In this case, MS ¹ analysis (for n = 1 the MS ⁿ analysis) ions called MS ¹ spectrum, select the MS ¹ spectrum, dissociated ions of MS ² spectra. Usually, after data acquisition, the data processor 6 confirms the MS ¹ spectrum data and the MS ² spectrum data, and performs valence determination.

FIG. 2 is an example of detection data of MS ⁿ spectrum in which a plurality of components are mixed in one mass at m / z in the data acquired in the liquid chromatograph mass spectrometer used in the embodiment of the present invention. is there. Further, FIG. 2 (a) is an example of MS spectrum data acquired by a mass spectrometer, and is bivalent (indicated as (2+) in the figure) that is not an isotope spectrum in an area of 1 mass at m / z. It is shown that the spectrum derived from the component of (2) and the spectrum derived from the component of trivalent (shown as (3+) in the figure) are adjacently mixed. In FIG. 2A, an accurate valence is displayed on the spectrum, but in the measured data, the adjacent spectrum is an isotope spectrum in a region where a plurality of adjacent components are mixed. May be misjudged. As a result, there is a possibility that an MS spectrum derived from a plurality of components may be simultaneously analyzed by MS ² , and an MS ² spectrum in which a plurality of components are mixed may be detected, which may complicate spectrum analysis. In addition, the location where the spectrum derived from a plurality of components adjoins indicates a location where a plurality of isotope spectra exist within 2 squares at m / z. Further, as a comparative example, FIG. 2B shows spectrum data when the valence is erroneously determined. This is because, unlike spectra with different valences as shown in FIG. 2 (a), bivalent and trivalent are accurately determined, but adjacent trivalent and bivalent areotope spectra. An example determined as (in this case, pentavalent (displayed as (5+) in the figure)) is shown.

  FIG. 3 is a flowchart of an analysis method for correcting the valence determination of an MS spectrum in acquired data in the liquid chromatograph mass spectrometer method according to the present invention. In this method, first, a process (step a) 7 for acquiring mass spectrometry data is performed by a mass spectrometer. Next, the valence determination process (step b) 8 of the mass spectrometry data is performed using the data processing unit. Next, a predetermined mixed region extraction process (step c) 9 extracts a region in which spectra derived from a plurality of components are mixed in the MS data. When the valence determination is performed in the valence determination process 8, the MS spectrum of the valence display that has been erroneously determined is still included.

Thereafter, the MS spectrum intensity and the MS ² spectrum intensity of the extracted mixed region are listed for each scan by the spectrum intensity list processing (step d) 10 for each scan. Next, the processing for calculating the inter-list variation for each scan (step e) 11 calculates the spectral intensity variation between the lists for each scan. Next, the group of spectra having the same variation is grouped by the peak group grouping process (step f) 12. Next, spectrum group integration processing (step g) 13 integrates all scans extracted from the intensity of the spectrum group used for grouping. Next, as a valence redetermination process (step h) 14, the data processor re-determines the valence based on the integrated spectrum group.

FIG. 4, FIG. 5 and FIG. 6 are examples showing a procedure for calculating the fluctuation of the total spectrum intensity for each scan in the MS ² spectrum data. That is, FIG. 4 is an example of a procedure for calculating the difference between the MS spectrum data and the MS ² spectrum data acquired in the liquid chromatograph mass spectrometer used in the embodiment of the present invention, and the change in the ratio. FIG. 5 is an example of a list of differences and ratios for each scan of MS spectrum data and MS ² spectrum data acquired in the liquid chromatograph mass spectrometer used in the embodiment of the present invention. Further, FIG. 6 is an example in which the spectrum fluctuations in FIG. 4 and FIG. 5 are listed for each scan, and then the grouped spectra are integrated. More specific description will be given below.

FIG. 4 is an MS ² spectrum of three consecutive scans. In (MS ² -1) to (MS ² -3), in order to calculate the difference for each scan, the first scan for each same mass number ( second scan from in FIG. (MS ² -1)) (in FIG. (MS ² -2), hereinafter the same. All spectral intensity difference), obtaining the total spectrum intensity difference 3 scans counted from the second scanning. Similarly, the total spectral intensity ratio from the first scan to the second scan is obtained for each same mass number, and the total spectral intensity ratio from the second scan to the third scan is obtained. If there are four or more consecutive scans, the same operation is repeated thereafter.

FIG. 5 is an example of a list of differences and ratios for each scan of MS spectrum data and MS ² spectrum data acquired in the liquid chromatograph mass spectrometer used in the embodiment of the present invention. It is the result of listing the spectral intensity difference and the spectral intensity ratio for each scan based on the spectral intensity.

First, in the list of differences for each scan in FIG. 5A, the spectrum b, the spectrum d, and the spectrum e show similar fluctuations in the difference. Further, the spectrum a, the spectrum c, and the spectrum f show the same difference variation. That is, in the spectrum b, the spectrum d, and the spectrum e, the spectrum intensity increases in (MS ² -1)-(MS ² -2) (here, the difference between the first scan and the second scan is negative). In (b), “−” is used for the notation. The same applies hereinafter.) And (MS ² -2)-(MS ² -3) indicate fluctuations in the decreasing direction (+). Furthermore spectrum a, the spectrum c, in the spectrum ^{f, (MS 2 -1) -} (MS 2 -2) in an increasing direction ^{(-), (MS 2 -2} ) - (MS 2 -3) even increasing direction (-) Shows fluctuations.

Also in the list of ratios for each scan in FIG. 5C, spectrum b, spectrum d, and spectrum e show similar ratio fluctuations, and spectrum a, spectrum c, and spectrum f show similar ratio fluctuations. ing. The Figure 5 (c), the spectrum b, spectral d, in the spectrum e, in ^{(MS 2 -1) / (MS} 2 -2) in ^{0.1, (MS 2 -2) /} (MS 2 -3) 5 shows the variation, spectrum a, the spectrum c, in the spectrum ^{f, (MS 2 -1) /} (MS 2 -2) in ^{30, (MS 2 -2) /} (MS 2 -3) 1.5 of the It shows the fluctuation. From the results of this list, a spectrum group in which the increase / decrease in the difference between scans and the intensity ratio show similar fluctuations, in this case, a group of spectrum b, spectrum d, and spectrum e, spectrum a, spectrum c, spectrum f, Each group is grouped. In FIGS. 5A to 5C, the spectrum b, the spectrum d, and the spectrum e are spectrum groups exhibiting similar fluctuations, and therefore, bold underlines are used to distinguish them from the spectrum a, the spectrum c, and the spectrum f. Indicated.

Then, what extracted the same group group and calculated the spectrum intensity is FIG. As shown in FIG. 6, all the scans extracted from the intensities of the spectrum groups used for grouping (in FIG. 6, (MS ² -1) to (MS ² -3), consecutive three scans of MS ² are shown). Spectrum), and the valence is re-determined with respect to the integrated spectrum group. In FIG. 6, the spectrum intensities from scan 1 to scan 3 of spectrum b, spectrum d, and spectrum e obtained by variability from the list of FIG. Then, the data processor 6 re-determines the valence based on the integrated spectrum. When the valence finally differs before and after the redetermination, the mass spectrometry data is corrected using the valence after the redetermination. Further, the fluctuation value obtained in the MS ² data is compared with the fluctuation value of only the MS data in the extraction range obtained in the same procedure, and the same fluctuation is obtained from the target MS spectrum and the MS spectrum. MS ² spectrum.

  In the above, an example of application to a liquid chromatograph mass spectrometer has been shown, but other various modifications can be made within the scope of the technical idea such as application to a gas chromatograph mass spectrometer.

An example of the disclosure of this specification is listed as follows.
(1) A mass spectrometry data analysis method for ionizing a sample to be measured, mass-analyzing the ions, detecting the ions, and analyzing the detected data, wherein the acquired MS ⁿ (n is n ≧ 1) In the spectral data, when analyzing the valence of the MS ⁿ spectrum, a mass spectrometric data analysis method that separates a plurality of mixed components and re-determines the valence.
(2) In (1) above,
a) MS ⁿ (n analyzing the ionized sample, selecting n ≧ 1 integer) spectral data, and the MS ⁿ spectrum as a target, obtaining a dissociated MS n ^{+ 1} spectral data,
b) determining the valence of the MS ⁿ spectrum data acquired in step (a) above;
c) extracting a predetermined region in the MS ⁿ spectrum data for which valence determination has been performed in advance in step (b) above;
d) listing MS ⁿ spectral data corresponding to the region extracted in step (c) and MS ^{n + 1} spectral data for each data acquisition scan;
calculating a variation of the spectral intensity for each e) MS ⁿ spectrum data for each said scan, and MS n ^{+ 1} spectral data,
f) grouping spectrum groups for each MS ⁿ spectral data and MS ^{n + 1} spectral data based on the variation in spectral intensity in step (e) above;
g) integrating the spectrum intensities of the extracted scans of the spectrum group grouped in step (f) above;
h) re-determining the valence for the MS ⁿ spectral data and the MS ^{n + 1} spectral data grouped and integrated in step (g),
A mass spectrometry data analysis method characterized in that MS ^{n + 1} spectrum data in which a plurality of components are mixed is grouped for each component by performing each of the above steps with respect to MS ⁿ spectrum data.
(3) In the above (1), in the step (e) and the step (f), the fluctuation of the spectral intensity difference for each scan is obtained, and the difference in the spectrum of the same mass number is either in the increasing direction or the decreasing direction. A method for analyzing mass spectrometry data, characterized by grouping a spectrum group that fluctuates in one direction.
(4) In the above (1), in the step (e) and the step (f), the fluctuation of the spectral intensity ratio for each scan is obtained, and the fluctuation of the spectral ratio of the same mass number is within a certain allowable range. A mass spectrometry data analysis method comprising grouping spectrum groups.
(5) In the above (1), in the step (e) and the step (f), both the difference for each scan and the fluctuation of the ratio are obtained, and the fluctuation of the spectrum of the same mass number is increased or decreased. A mass spectrometry data analysis method characterized by grouping a spectrum group which fluctuates in either one and whose fluctuation is within a certain allowable range.
(6) The mass spectrometry data analysis method according to (1), further including a step of determining the valence of the parent ion based on the step (h).
(7) A mass spectrometry data analysis method according to the above (1), wherein in step (d), a change in spectral intensity is obtained for at least three scans.
(8) Using a mass spectrometer having an ion source, a mass analyzer, a detector, and a data processor, an object to be measured is ionized by the ion source, and the ions are analyzed by the mass analyzer. A mass analysis data analysis method for detecting ions by the detection unit and analyzing the data acquired by the data processing unit, a) MS ⁿ in which an ionized sample is analyzed by a mass spectrometer
(N is, n ≧ 1 integer) selected spectral data, and the MS ⁿ spectrum as a target, obtaining a dissociated MS n ^{+ 1} spectral data, b) MS ⁿ spectra acquired in step (a) A step of performing valence determination on data in a data processing unit of the mass spectrometer; c) a step of extracting a predetermined mixed region in the MS ⁿ spectrum data subjected to valence determination in advance in step (b) above; d) listing MS ⁿ spectral data and MS ^{n + 1} spectral data corresponding to the mixed region extracted in step (c) for each data acquisition scan; and e) MS ⁿ spectral data for each scan. And calculating the variation in total spectral intensity for each MS ^{n + 1} spectral data;
f) grouping spectrum groups for each MS ⁿ spectral data and MS ^{n + 1} spectral data having a certain fluctuation based on the fluctuation of the spectral intensity in the step (e), The step of integrating the spectral intensities of the extracted scans of the spectrum group grouped in f) and the MS ⁿ spectrum data and the MS ^{n + 1} spectrum data grouped and integrated in the above step (g) and a step of re-determination of the, for MS ⁿ spectrum data, by performing the above steps, mass spectrometry, wherein the grouping MS n ^{+ 1} spectral data more components are mixed for each component Data analysis method.

As described above, in the mass spectrometer acquisition data, in a region where MS ⁿ spectra derived from a plurality of components are adjacently mixed, there is a possibility that MS ^{n + 1} analysis of the adjacent MS ⁿ spectrum may be performed simultaneously. May be complicated. Therefore, more reliable data is provided by re-determining the valence of the MS ^{n + 1} (n is an integer of n ≧ 1) spectrum and the MS ⁿ spectrum. That is, a region where the MS ^{n + 1} spectrum is mixed adjacently is extracted, and a region where valence re-determination is performed is determined. Thereafter, the extracted spectrum intensities of the predetermined mixed region are listed for each scan, the spectrum intensity fluctuation between the lists for each scan is calculated, and the group of spectra having the same fluctuation is grouped. It is disclosed to perform analysis of mass spectrometry data including a step of re-determination of valence based on the grouped data.

  In the present specification, the mass analyzer 4 may be an independent single mass spectrometer or a mass spectrometer built in a liquid chromatograph mass spectrometer.

It is the schematic of the flow of the whole liquid chromatograph mass spectrometry method used in the Example of this invention. It is an example of the detection data of the MS ⁿ spectrum in which a plurality of components are mixed in one mass in the data acquired in the liquid chromatograph mass spectrometer used in the example of the present invention. It is an example of the operation procedure at the time of analyzing ^MSn spectrum data and MSn ^{+ 1} spectrum data in the example of the present invention. It is an example of a procedure for calculating MS spectrum data obtained in a liquid chromatograph mass spectrometer used in the embodiment of the present invention, and the difference between the MS ² spectral data, and the variation of the ratio. Examples MS spectrum data acquired in a liquid chromatograph mass spectrometer to be used, and MS ² difference in each scan of the spectral data of the present invention, and is an example of a list of ratios. FIG. 4 and FIG. 5 show an example in which spectrum fluctuations are listed for each scan and then the grouped spectra are integrated.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Sample 2 Liquid chromatograph 3 Ion source 4 Mass analysis part 5 Detection part 6 Data processing part 7 Acquisition of mass spectrometry data (step a)
8 Valency determination of the data (step b)
9 Extraction of predetermined mixed area (step c)
10 List of spectral intensities for each scan (step d)
11 Calculation of spectral intensity fluctuation for each scan (step e)
12 Group of spectra (step f)
13 Integration of grouped spectra (step g)
14 Re-determination of valence (step h)

Claims (7)

A mass spectrometry data analysis method for ionizing a sample to be measured, mass analyzing the ions, detecting the ions, and analyzing the detection data,
a) MS ⁿ (n analyzing the ionized sample, selecting n ≧ 1 integer) spectral data, and the MS ⁿ spectrum as a target, obtaining a dissociated MS n ^{+ 1} spectral data,
b) determining the valence of the MS ⁿ spectrum data acquired in step (a) above;
c) extracting a predetermined region in the MS ⁿ spectrum data for which valence determination has been performed in advance in step (b) above;
d) listing MS ⁿ spectral data corresponding to the region extracted in step (c) and MS ^{n + 1} spectral data for each data acquisition scan;
calculating a variation of the spectral intensity for each e) MS ⁿ spectrum data for each said scan, and MS n ^{+ 1} spectral data,
f) grouping spectrum groups for each MS ⁿ spectral data and MS ^{n + 1} spectral data based on the variation in spectral intensity in step (e) above;
g) integrating the spectrum intensities of the extracted scans of the spectrum group grouped in step (f) above;
h) re-determining the valence for the MS ⁿ spectral data and the MS ^{n + 1} spectral data grouped and integrated in step (g),
A mass spectrometry data analysis method characterized in that MS ^{n + 1} spectrum data in which a plurality of components are mixed is grouped for each component by performing each of the above steps with respect to MS ⁿ spectrum data.   In claim 1, in steps (e) and (f), a change in spectral intensity difference for each scan is obtained, and a difference in spectrum of the same mass number fluctuates in either an increasing direction or a decreasing direction. Mass spectrometry data analysis method, characterized in that spectrum groups to be grouped are grouped.   In claim 1, in steps (e) and (f), a change in spectral intensity ratio for each scan is obtained, and a group of spectra in which the fluctuation in the spectral ratio of the same mass number is within a certain allowable range is grouped. A method for analyzing mass spectrometry data, characterized by comprising:   In claim 1, in the step (e) and the step (f), both the difference for each scan and the fluctuation of the ratio are obtained, and the fluctuation of the spectrum of the same mass number is in either the increasing direction or the decreasing direction. A method for analyzing mass spectrometry data, characterized by grouping a spectrum group that fluctuates and whose ratio fluctuation is within a certain allowable range.   The mass spectrometry data analysis method according to claim 1, further comprising a step of performing valence determination of a parent ion based on the step (h).   2. The method of analyzing mass spectrometry data according to claim 1, wherein in step (d), a change in spectral intensity is determined for at least three scans. Using a mass spectrometer having an ion source, a mass analyzer, a detector, and a data processor, the object to be measured is ionized by the ion source, the ions are analyzed by the mass analyzer, and the detection is performed. A mass spectrometry data analysis method for detecting ions in a unit and analyzing the data acquired in the data processing unit,
MS ⁿ (n analyzing the ionized sample from a) mass spectrometer, selecting n ≧ 1 integer) spectral data, and the MS ⁿ spectrum as a target, obtaining a dissociated MS n ^{+ 1} spectral data ,
b) performing the valence determination on the MS ⁿ spectrum data acquired in step (a) in the data processing unit of the mass spectrometer;
c) extracting a predetermined mixed region in the MS ⁿ spectrum data subjected to valence determination in advance in the above step (b);
d) listing MS ⁿ spectral data corresponding to the mixed region extracted in step (c) and MS ^{n + 1} spectral data for each data acquisition scan;
e) calculating the variation in total spectral intensity for each MS ⁿ spectral data and MS ^{n + 1} spectral data for each scan;
f) based on the variation of the spectral intensity of step (e), the steps of grouping the spectral groups MS ⁿ spectrum data that a certain variation in, and every MS n ^{+ 1} spectral data,
g) integrating the spectrum intensities of the extracted scans of the spectrum group grouped in step (f) above;
h) re-determining the valence for the MS ⁿ spectral data and the MS ^{n + 1} spectral data grouped and integrated in step (g),
A mass spectrometry data analysis method characterized in that MS ^{n + 1} spectrum data in which a plurality of components are mixed is grouped for each component by performing each of the above steps with respect to MS ⁿ spectrum data.

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